RESUMEN

Biodesulfurization is a promising alternative for removing sulfur molecules from the polycyclic aromatic sulfur compounds (PASC) found in petroleum. PASC consists of recalcitrant molecules that can degrade fuel quality and cause a range of health and environmental problems. Therefore, identifying bacteria capable of degrading PASC is essential for handling these recalcitrant molecules. Microorganisms in environments exposed to petroleum derivatives have evolved specific enzymatic machinery, such as the 4S pathway associated with the dszABC genes, which are directly linked to sulfur removal and utilization as nutrient sources in the biodesulfurization process. In this study, bacteria were isolated from a bioreactor containing landfarm soil that had been periodically fed with petroleum for 12 years, using a medium containing dibenzothiophene (DBT), 4.6-dimethylbenzothiophene, 4-methylbenzothiophene, or benzothiophene. This study aimed to identify microorganisms capable of degrading PASC in such environments. Among the 20 colonies isolated from an inoculum containing DBT as the sole sulfur source, only four isolates exhibited amplification of the dszA gene in the dszABC operon. The production of 2-hydroxybiphenyl (HPB) and a decrease in DBT were detected during the growth curve and resting cell assays. The isolates were identified using 16S rRNA sequencing belonging to the genera Stutzerimonas and Pseudomonas. These isolates demonstrated significant potential for biodesulfurization and/or degradation of PASC. All isolates possessed the potential to be utilized in the biotechnological processes of biodesulfurization and degradation of recalcitrant PASC molecules.

Asunto(s)

Petróleo , Compuestos Policíclicos , Compuestos de Azufre , ARN Ribosómico 16S/genética , Azufre , Reactores Biológicos , Bacterias/genética

RESUMEN

Traditional therapy for malignant neoplasms involving surgical procedures, radiotherapy and chemotherapy aims to kill neoplastic cells, but also affects normal cells. Therefore, exogenous proteases are the target of studies in cancer therapy, as they have been shown to be effective in suppressing tumors and reducing metastases. Pluronic F127 (F127) is a copolymer of amphiphilic blocks that has shown significant potential for drug administration, as it is capable of incorporating hydrophobic drugs and self-assembling in micrometers of nanometric size. This study investigated the effects of immobilization of the alkaline protease PR4A3 with pluronic F127 micelles on the enzyme-induced cytotoxicity. Protease immobilization was demonstrated through UV-visible and circular dichroism (CD) spectroscopies, as the enzyme interacts with the polymeric micelle of Pluronic F127 without changing its secondary structure. In addition, the immobilized form of the enzyme showed greater bioavailability after passing through the simulated gastrointestinal transit. Cell viability was assessed using the tetrazoic methylthiazole (MTT) assay. The results open perspectives for new research and development for PR4A3 in the treatment of colorectal carcinoma.

Asunto(s)

Adenocarcinoma/patología , Proteínas Bacterianas/metabolismo , Neoplasias Colorrectales/patología , Endopeptidasas/metabolismo , Enzimas Inmovilizadas/metabolismo , Micelas , Poloxámero/química , Disponibilidad Biológica , Muerte Celular , Línea Celular Tumoral , Dicroismo Circular , Tránsito Gastrointestinal , Humanos , Hidrólisis , Proteolisis , Espectrofotometría Ultravioleta

RESUMEN

In the present work we aimed to demonstrate the influence of inoculum starter in support high quality fermentation. Cocoa fermentations were performed in wooden boxes and eight yeasts strains were used in separated fermentations of fine cocoa, type Scavina, as starter inoculum. Temperature, pH, titirable acidity, reducing sugar and free amino acids were evaluated during or after fermentation. The influence of starters yeasts on the decrease of acidity, sugar concentration and free amino acids was significant. The strains Candida parapsilosis, Torulaspora delbrueckii and Pichia kluyveri showed greater changes in the reducing sugar and free amino acids in fermented cocoa beans. These results indicate the ability of yeast used as inoculum starter to modify the end condition and further enhance the quality of fine cocoa beans.

Asunto(s)

Cacao , Microbiología de Alimentos/métodos , Levaduras , Aminoácidos/análisis , Aminoácidos/metabolismo , Cacao/química , Cacao/metabolismo , Candida parapsilosis/genética , Candida parapsilosis/metabolismo , Chocolate , Fermentación , Concentración de Iones de Hidrógeno , Pichia/genética , Pichia/metabolismo , Semillas/química , Semillas/microbiología , Temperatura , Torulaspora/genética , Torulaspora/metabolismo , Levaduras/genética , Levaduras/metabolismo

RESUMEN

Many synthetic biologists have adopted methods based on Type IIS restriction enzymes and Golden Gate technology in their cloning procedures, as these enable the combinatorial assembly of modular elements in a very efficient way following standard rules. GoldenBraid (GB) is a Golden Gate-based modular cloning system that, in addition, facilitates the engineering of large multigene constructs and the exchange of DNA parts as result of its iterative cloning scheme. GB was initially developed specifically for plant synthetic biology, and it has been subsequently extended and adapted to other organisms such as Saccharomyces cerevisiae, filamentous fungi, and human cells by incorporating a number of host-specific features into its basic scheme. Here we describe the general GB cloning procedure and provide detailed protocols for its adaptation to filamentous fungi-a GB variant known as FungalBraid. The assembly of a cassette for gene disruption by homologous recombination, a fungal-specific extension of the GB utility, is also shown. Development of FungalBraid was relatively straightforward, as both plants and fungi can be engineered using the same binary plasmids via Agrobacterium-mediated transformation. We also describe the use of a set of web-based tools available at the GB website that assist users in all cloning procedures. The availability of plant and fungal versions of GB will facilitate genetic engineering in these industrially relevant organisms. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Software-assisted modular DNA assembly of a two gene expression-cassette with GB Basic Protocol 2: Agrobacterium tumefaciens-mediated transformation of filamentous fungi Basic Protocol 3: Software-assisted modular DNA assembly of a gene disruption-cassette using GB Basic Protocol 4: Obtaining disruption transformants.

Asunto(s)

Clonación Molecular/métodos , Hongos/genética , Ingeniería Genética/métodos , Plantas/genética , Secuencia de Bases , ADN/genética , Enzimas de Restricción del ADN/metabolismo , Expresión Génica , Vectores Genéticos , Células HEK293 , Humanos , Plásmidos/genética , Biología Sintética/métodos

RESUMEN

Microorganisms native to mangroves are expected to contain enzymes capable of hydrolyzing different carbon sources. However, most of these microorganisms aren't cultivable; hence, alternative techniques as metagenomics are tools for studying and obtaining some of the natural genomes, genes and enzymes of biotechnological interest. The ß-glucanase was produced using a metagenomic clone of mangrove sediments and detected by functional screening on carboxymethylcellulose substrate. The enzyme was purified by cation exchange chromatography. The peptides detected by mass spectrometry showed 20% identity with the polypeptide deduced from the genomic fragment sequenced. The ORF identified as BglfosD9 possessed 729 bp and the encoded protein showed predicted MW and pI of 28kD and 6.8, respectively. The enzyme was active in a wide range of pH (5-10) with optimum pH at 8, had relative activity greater than 50% at all temperatures tested (5-90 °C), was stable at temperatures of 5, 50 and 90 °C and showed excellent relative activity at high NaCl concentrations. This ß-glucanase also showed high relative activity in the presence of SDS and it could hydrolyze ß-glucan, CMC and Avicel as substrates. These findings support the idea of a new thermostable and active enzyme at basic pH from metagenomic library of mangrove sediment.

Asunto(s)

Bacillus , Sedimentos Geológicos/microbiología , Glicósido Hidrolasas , Humedales , Bacillus/enzimología , Bacillus/genética , Carboximetilcelulosa de Sodio/química , Clonación Molecular , Estabilidad de Enzimas , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Calor , Metagenoma , Salinidad

RESUMEN

The mineralogical composition of caves makes the environment ideal for inhabitation by microbes. However, the bacterial diversity in the cave ecosystem remains largely unexplored. In this paper, we described the bacterial community in an oxic chamber of the Sopradeira cave, an iron-rich limestone cave, in the semiarid region of Northeast Brazil. The microbial population in the cave samples was studied by 16S rDNA next-generation sequencing. A type of purple sulfur bacteria (PSB), Chromatiales, was found to be the most abundant in the sediment (57%), gravel-like (73%), and rock samples (96%). The predominant PSB detected were Ectothiorhodospiraceae, Chromatiaceae, and Woeseiaceae. We identified the PSB in a permanently aphotic zone, with no sulfur detected by energy-dispersive X-ray (EDX) spectroscopy. The absence of light prompted us to investigate for possible nitrogen fixing (nifH) and ammonia oxidizing (amoA) genes in the microbial samples. The nifH gene was found to be present in higher copy numbers than the bacterial-amoA and archaeal-amoA genes, and archaeal-amoA dominated the ammonia-oxidizing community. Although PSB dominated the bacterial community in the samples and may be related to both nitrogen-fixing and ammonia oxidizing bacteria, nitrogen-fixing associated gene was the most detected in those samples, especially in the rock. The present work demonstrates that this cave is an interesting hotspot for the study of ammonia-oxidizing archaea and aphotic PSB.

RESUMEN

Restricted contact with the external environment has allowed the development of microbial communities adapted to the oligotrophy of caves. However, nutrients can be transported to caves by drip water and affect the microbial communities inside the cave. To evaluate the influence of aromatic compounds carried by drip water on the microbial community, two limestone caves were selected in Brazil. Drip-water-saturated and unsaturated sediment, and dripping water itself, were collected from each cave and bacterial 16S rDNA amplicon sequencing and denaturing gradient gel electrophoresis (DGGE) of naphthalene dioxygenase (ndo) genes were performed. Energy-dispersive X-ray spectroscopy (EDX) and atomic absorption spectroscopy (AAS) were performed to evaluate inorganic nutrients, and GC was performed to estimate aromatic compounds in the samples. The high frequency of Sphingomonadaceae in drip water samples indicates the presence of aromatic hydrocarbon-degrading bacteria. This finding was consistent with the detection of naphthalene and acenaphthene and the presence of ndo genes in drip-water-related samples. The aromatic compounds, aromatic hydrocarbon-degrading bacteria and 16S rDNA sequencing indicate that aromatic compounds may be one of the sources of energy and carbon to the system and the drip-water-associated bacterial community contains several potentially aromatic hydrocarbon-degrading bacteria. To the best of our knowledge, this is the first work to present compelling evidence for the presence of aromatic hydrocarbon-degrading bacteria in cave drip water.

RESUMEN

Functional screening of metagenomic libraries is an important tool for the discovery of new molecules. The metabolic diversity of microorganisms enables survival in harsh environments and is related to the production of enzymes. In this study, we identified a protease-producing clone from a metagenomic library derived from mangrove sediment. The protease was purified by ammonium sulphate precipitation and gel filtration chromatography, with a yield of 77.27% and a specific activity of 8.57 U µg-1 . It had a molecular weight of approximately 70 kDa. MS/MS in ESI-Q-TOF revealed nine peptides similar to a peptidase of Bacillus safensis. The aligned partial sequence showed 47.48% identity and 82.74% similarity to the conserved domains of a glutamyl aminopeptidase from the human gut metagenome and 32.12% total coverage. The protease had an optimal pH of 8.5 and optimal activity at 60°C. At pH 9-12, its activity was greater than 80%. It had moderate thermotolerance and thermostability at temperatures of 40 and 50 °C. The KM and Vmax values were estimated to be 0.92 mg ml-1 , and 13.15 mmol min-1 for azocasein. Substrate specificity analysis showed that PR4A3 was active on gelatin, blood, egg yolk, and milk. These results support the potential use of PR4A3 in biotechnological applications.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Endopeptidasas/química , Endopeptidasas/metabolismo , Sedimentos Geológicos/microbiología , Metagenómica , Humedales , Secuencia de Aminoácidos , Bacillus/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Biotecnología , Brasil , Cromatografía en Gel , Endopeptidasas/genética , Endopeptidasas/aislamiento & purificación , Activación Enzimática , Pruebas de Enzimas , Estabilidad de Enzimas , Calor , Concentración de Iones de Hidrógeno , Cinética , Metagenoma , Peso Molecular , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Péptido Hidrolasas/aislamiento & purificación , Sales (Química) , Alineación de Secuencia , Especificidad por Sustrato , Espectrometría de Masas en Tándem , Temperatura

RESUMEN

BACKGROUND: The presence of organic sulfur-containing compounds in the environment is harmful to animals and human health. The combustion of these compounds in fossil fuels tends to release sulfur dioxide in the atmosphere, which leads to acid rain, corrosion, damage to crops, and an array of other problems. The process of biodesulfurization rationally exploits the ability of certain microorganisms in the removal of sulfur prior to fuel burning, without loss of calorific value. In this sense, we hypothesized that bacterial isolates from tropical landfarm soils can demonstrate the ability to degrade dibenzothiophene (DBT), the major sulfur-containing compound present in fuels. RESULTS: Nine bacterial isolates previously obtained from a tropical landfarm soil were tested for their ability to degrade dibenzothiophene (DBT). An isolate labeled as RR-3 has shown the best performance and was further characterized in the present study. Based on physiological aspects and 16 s rDNA sequencing, this isolate was found to be very closely related to the Bacillus pumillus species. During its growth, high levels of DBT were removed in the first 24 hours, and a rapid DBT degradation within the first hour of incubation was observed when resting cells were used. Detection of 2-hydroxybiphenyl (HBP), a marker for the 4S pathway, suggests this strain has metabolical capability for DBT desulfurization. The presence of MgSO4 in growth medium as an additional sulfur source has interfered with DBT degradation. CONCLUSIONS: To our knowledge, this is the first study showing that a Bacillus strain can metabolize DBT via the 4S pathway. However, further evidences suggest RR-3 can also use DBT (and/or its derivative metabolites) as carbon/sulfur source through another type of metabolism. Compared to other reported DBT-degrading strains, the RR-3 isolate showed the highest capacity for DBT degradation ever described in quantitative terms. The potential application of this isolate for the biodesulfurization of this sulfur-containing compound in fuels prior to combustion was discussed.

Asunto(s)

Bacillus/aislamiento & purificación , Bacillus/metabolismo , Microbiología del Suelo , Compuestos de Azufre/metabolismo , Tiofenos/metabolismo , Bacillus/clasificación , Bacillus/genética , Biotransformación , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Redes y Vías Metabólicas , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Factores de Tiempo , Clima Tropical

RESUMEN

Anomalies of the flexor digitorum superficialis muscle are extremely uncommon and usually present as a painful mass or pseudotumour within the palm. Diagnosis may be difficult because many other soft tissue tumours (lipomas, ganglions, giant cell tumours and hamartomas) may present similarly. Magnetic resonance imaging helps to define the extent and characteristics of this anomalous muscle belly and to distinguish it from a soft tissue sarcoma, whereas plain radiographs are of little value. Three types of flexor digitorum superficialis muscle anomalies have been described, and treatment consists of subtotal or total surgical debulking of the mass if symptoms persist or if the diagnosis is in question. Most patients have complete resolution and full recovery. To date, 20 cases have been reported in the literature, usually involving the right small finger. In the present paper, the case of an anomalous flexor digitorum superficialis muscle in a 17-year-old male patient's left index finger is reported. Symptoms were relieved following surgical debulking and hand-based occupational therapy.